Sterile sodium chloride solution 0.9% in water (S) and the antiseptic solution (AS) of chlorhexidine digluconate 10mg/mL were purchased from a local pharmacy (Sao Paulo Pharmacy – Ribeirao Preto, Brazil). H. brasiliensis SLX was obtained from Pelenova Biotecnologia S/A Company (Distrito Industrial – Ribeirao Preto, Brazil) as described by Mendonça.16 Briefly, the latex was obtained from a rubber tree (H. brasiliensis) of the clone RRhim 600. Natural latex was treated with ammonium hydroxide (2%) when collected so that coagulation was avoided. A 2.2% acetic acid solution (1:2 v/v) was added under gentle stirring. In a next step, the pure serum was separated from the rubber and subjected to dialysis and lyophilization. The purification and characterization of the serum were performed as previously described to Mendonça17 using two-dimensional electrophoresis and mass spectrometry. Three main fractions were obtained, and named FrHB1, FrHB2, and FrHB3. Further, according the manufacturer of final product (gel-cream) used in pre-clinical experiments and also in clinical use, to eliminate the protein causing the greatest number of allergic reactions, hevein and its derivatives, the process of tangential filtration was performed to consider only substances below 10kDa. Pure SLX was diluted in Dulbecco's Modified Eagle Medium (DMEM) at concentrations of 0.1–1% for the viability assay and 0.00001–1% for the migration/proliferation assay. For in vivo experiments, SLX in gel-cream was used (Valeant Pharmaceuticals International Inc. – São Paulo, SP, Brazil).

NIH-3T3 (mouse fibroblast cell line) cells were purchased from the American Type Culture Collection (ATCC CRL-1658; Manassas – Virginia, United States). Human keratinocytes were isolated from healthy fragments of human skin with the informed consent of patients undergoing either breast or abdomen plastic surgery at the Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil. All protocols were approved by the Research Ethics Committee of Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil – process 5606/2008.

Fibroblasts were cultured in high-glucose DMEM (4.5g/L; GIBCO – Invitrogen Corporation; Grand Island, NY, United States) and keratinocytes in Defined Keratinocyte Medium (GIBCO – Invitrogen Corporation; Grand Island, NY, United States) and were both supplemented with 10% fetal bovine serum and 1% antibiotic and antimycotic (GIBCO – Invitrogen Corporation; Grand Island, NY, United States), at 37°C, in a humidified atmosphere containing 5% CO2.

The viability of NIH-3T3 fibroblasts and human keratinocytes was determined by tetrazolium 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-bromide MTT colorimetric assay.18 Fibroblasts and keratinocytes were seeded in 96-well microplates at 2×104cells/mL concentration. Plates were incubated overnight for cellular adhesion. After this time, the supernatant was aspirated and 200μL of test solutions containing SLX were added according to the following concentrations: only basal cell culture medium (positive control), 0.1% and 1% (SLX concentration was diluted in culture medium) and 50%/50% of culture medium and dimethyl sulfoxide (DMSO as negative control). Plates were incubated for 24h and 48h. After incubation, the supernatant was aspirated and the cells were washed with phosphate-buffered saline (PBS). Then, 20μL of the stock solution of MTT (Sigma-Aldrich Co. LLC; 5mg/mL in PBS) in 180μL DMEM culture medium (without phenol red) was added to each well, and the plates were incubated under the same conditions for an additional 3h. After that, the MTT solution was removed, and 200μL of DMSO was added to dissolve the formazan crystals. Absorbance was read at 570nm. The experiment was performed in triplicate, considering three independent experiments. The results were expressed as a percentage of cell viability.18–20

To analyze the proprieties of SLX that stimulate keratinocyte migration/proliferation, an in vitro scratch assay was used.21,22 Human keratinocytes were seeded in 24-well culture plates at a concentration of 3×104cells/well and were cultured with medium containing 10% fetal bovine serum until reaching a confluence of approximately 80%. Then, a linear scratch, mimicking an artificial wound, was created in the cellular monolayer using a 200μL plastic tip. The medium was removed, and the cells were washed with PBS. Medium with 50% DMSO (negative control), culture medium with 10% fetal bovine serum (basal), set to 0 on the X-axis, and mediumS containing 1%, 0.1%, 0.01%, 0.001%, 0.0001%, or 0.00001% SLX concentrations were added in three wells per group and were incubated for 24h at 5% CO2 at 37°C. After 24h, the wells were washed with PBS and were fixed with 4% paraformaldehyde for 15min. The cells were washed again and stained with 4′,6-diamino-2-phenyl-indole (DAPI) for 5min. Images of scraped areas were acquired using a fluorescence microscope coupled with a camera (Carl Zeiss® – Oberkochen, GE). The proliferation/migration of cells into the scraped region was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, United States). The results were expressed as a percentage of cells that proliferate and/or migrate into the scratched area after SLX treatment compared to the number of migration/proliferation cells in control group. The experiments were also carried out in triplicate, considering also three independent experiments.

Studies were conducted and approved by the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation (COBEA) and approved by the Ethical Commission of Ethics in Animal Research (CETEA) – Ribeirão Preto Medical School, University of São Paulo, registry No. 072/2012.

A total of 72 adult male Wistar rats (Rattus norvegicus, 180–200g) from the Central Animal Facility of Ribeirão Preto Medical School, São Paulo, Brazil, were housed for seven days before experimental process. During the entire experimental period, the rats were maintained in individual polycarbonate cages at a constant temperature (23±2°C) and humidity (55%) under a 12/12h light/dark cycle. The animals had free access to a standard chow diet and drinking water.

Rats were anesthetized intraperitoneally with ketamine (70mg/kg) and xylazine (12mg/kg). The dorsum cervical region of each rat was trichotomized and cleaned with 70% alcohol. A dermoabrasor LB-100 device (BELTEC Indústria e Comércio de Equipamentos Odontológicos – Araraquara, SP, Brazil) was used to perform the excoriations with a diamond sanding disk RH14633 (RHOSSE Instrumentos e Equipamentos Cirúrgicos – Ribeirão Preto, SP, Brazil) on the back of the animal with a cutaneous lesion of approximately 2cm2. Intraperitoneal dipyrone, at 50mg/kg of body weight, was administered diluted in saline two times a day (12/12h) during the first 48h for preventing pain. Then, the animals were allocated in individual cages until the end of the experiment. The animals were divided into three groups, with 24 rats each, and treated daily with SLX 1%, a standard antiseptic solution (AS) (chlorhexidine digluconate 10mg/mL) as a positive control, and saline (S) for ten days, without occlusive dressing.

Images of the abrasion wounds were obtained on days 0, 2, 7, and 10 after treatment using a 14-megapixel digital camera (Kodak EasyShare M575) that was fixed in a standardized support for imaging, presenting a ruler as measurement reference and the calculation of the abrasion healing rate (AHR) was made by the following formula [(initial area−final area)/initial area] as described and performed by Leite23 for cutaneous wounds.

Eight animals from each group were euthanized using an excessive dose of anesthetic on days 2, 7, and 10. On each day of follow-up, a fragment was harvested with scissors. Half of the sample was embedded in 10% buffered formaldehyde solution (v/v) for histological study (hematoxylin–eosin); the other part of the sample wound was conditioned at −80°C for biochemical assays.

Biopsies were embedded in paraffin as previously described Andrade.24 A Leica DM 4000B optical microscope equipped with a LEICA DFC 280 camera (Leica Microsystems® – Germany) was used to capture the histological images at 400× magnification, using Leica Application Suite (LAS) software v. 3.2.0. One complete cut section of the lesion areas per animal (transversal section) was performed, in which three images were taken; three measurements were performed in each image. Both the measurements of the crust and epidermis were performed from one end to the other, mainly based on the neutrophil inflammatory infiltrate into the edge of epidermis. The image was opened and a line was drawn from the granular layer of the epidermis until the transition with the dermis (thickness of the epidermis) and thickness of the crust. At the end of the tracing, the software provided the distance in μm.25

The harvested biopsies were conditioned in 2mL plastic tubes with 200μL 0.1M NaCl buffer, 0.02M NaPO4, 0.015M NaEDTA pH 4.7, and remained at −70°C until use. The fragments were homogenized by an Omni Tissue Homogenizer (Kennesaw, GA, United States) at 13,000rpm. After centrifugation, the fragments were resuspended in NaPO4 buffer (pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide (HTAB). Then, 5μL of the supernatant from the samples were placed in a 96-well plate for the assay. A standard curve for the neutrophils was obtained with isolated peritoneal mice neutrophils after 6h of 0.25mL carrageenan 1% (Sigma Aldrich) inoculation into the peritoneal cavity of the mice. Twenty-five microliters of 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma Chemical Company – St. Louis, MO, United States) was added to each well of the plate (samples and standard curve of the neutrophils), followed by 100μL of H2O2. The reaction was then stopped with 4M sulfuric acid and read on a plate reader at 450nm.26

Data was expressed as the mean value±standard error of mean (SEM). Statistical analysis was performed using GraphPad software (San Diego, CA, United States). Statistical variations between groups were determined using one-way ANOVA analysis (variance for multiple comparisons) followed by a Bonferroni post-test. Values of p<0.05 were considered significant.

Initially, the in vitro viability of SLX was evaluated by the MTT colorimetric method using the NIH-3T3 fibroblast cell line and the human keratinocytes. After 24h of incubation, SLX 1% and 0.1% did not exhibit any cytotoxic effects against fibroblasts compared to basal control (only cell culture medium, considered as 100% viability), showing 109% and 105% viability, respectively (Fig. 1A). Similar results were observed with human keratinocytes exhibiting 106% and 95% viable cells after SLX 1% and 0.1% exposure, respectively (Fig. 1B).

Figure 1.

Cell viability. Percentage of viability of NIH-3T3 (undefined) fibroblasts (A–C) and human keratinocytes (B–D) in culture at 24h (A and B) and 48h (C and D) relative to the control culture (corresponding to 100% viable cells) by the MTT method, distributed at 1% and 0.1% concentrations of latex serum. Values represent means±standard error of mean (SEM) of triplicate results. ANOVA statistical test, Bonferroni post-test. *Significant difference (p<0.05) in relation to the negative control. #Significant difference (p<0.05) between control groups.

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After 48h of incubation, similar results were observed. Fibroblasts showed viability percentages of 91% and 94% for 1% and 0.1% of SLX, respectively (Fig. 1C), and 119% and 111%, for keratinocytes, respectively (Fig. 1D).

To evaluate in vitro cell migration/proliferation, the scratch assay was performed using human keratinocytes. After 24h, SLX showed dose dependence increased in human keratinocyte proliferation/migration. The highest stimulatory effect was observed with 0.1% and 0.01% concentrations, and enhanced cell numbers by up to 70%. 1% SLX exhibited an antiproliferative effect (p<0.05) as compared to the number of cells in the control group (Fig. 2A and B).

Figure 2.

Cell migration. (A) Microscopic fluorescent 4′,6-diamino-2-phenyl-indole (DAPI) image of the scratch assay with human keratinocytes. Cell proliferation/migration in the scratch assay was observed in response to an “artificial lesion” treated with different concentrations of latex serum. (B) Percentage of proliferation/migration of human keratinocytes relative to basal culture medium, set to 0 on the X-axis, distributed at various concentrations of latex serum (SLX) treated for 24h in culture. Values represent mean±standard error of mean (SEM) of triplicate results. ANOVA statistical test, Bonferroni post-test. *Corresponds to a significant difference (p<0.05) between groups (magnification: 50×).

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The re-epithelialization process was evaluated by means of a scarring index of abrasions. On the 2nd day of follow-up, all groups that were studied presented similar re-epithelialization (p>0.05). However, on the 7th day, the SLX group presented a higher re-epithelialization rate compared to the S and AS groups (p<0.05). On the 10th day, the abrasions were almost re-epithelized, reaching an average percentage of 83% in the S group, 88% in the AS group, and 99% in the SLX Group (p>0.05) (Fig. 3A and B).

Figure 3.

Re-epithelialization. (A) Clinical follow-up of cutaneous abrasions. (B) Quantification of re-epithelialization (by the abrasion healing rate [AHR]) treated with saline (S), antiseptic solution (AS), and latex serum (SLX). ANOVA statistical test, Bonferroni post-test. *Corresponds to a significant difference (p<0.05).

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Hematoxylin and eosin staining was used for histological analysis. Fig. 4 presents representative images (images with smaller and larger amounts of crust and epidermis were chosen) during different aspects of re-epithelialization of the groups on the 10th day. The SLX group highlighted the smaller amount of crust and a very thick epidermis, with a greater number of layers of keratinocytes in contrast with other groups that still presented a thicker crust and thinner epidermis.

Figure 4.

Photomicrography of the exulcerated areas. Photomicrography of the exulcerated areas treated topically with saline (S), antiseptic solution (AS), or latex serum (SLX) on the 10th day of follow-up; samples were stained with hematoxylin–eosin (HE), highlighting the amount of crust and the thickness of the epidermis with a worse and a better photomicrograph in each group and treatment. The red arrows indicate the thickness of the epidermis (magnification: 100×).

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Regarding crust thickness, the SLX Group showed a smaller crust thickness than those of the S Group. The AS Group also showed a smaller crust thickness than the S Group (p<0.05; Fig. 5A). The SLX Group had the thickest epidermis among the groups (p<0.05; Fig. 5B).

Figure 5.

Distribution of the mean thickness (μm) of the crust and epidermis. (A) Distribution of the mean thickness (μm) of the crust and (B) distribution of the mean thickness (μm) of the epidermis of the groups treated with saline (S), antiseptic solution (AS), or latex serum (SLX) on the 10th day of the follow-up. Values represent mean±standard error of mean (SEM). ANOVA statistical test, Bonferroni post-test. *Corresponds to a significant difference (p<0.05).

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The evaluation of neutrophil infiltration during the inflammatory process was estimated by myeloperoxidase content in the abrasion biopsies (n=8 per group/day). A high concentration of MPO was observed in all groups on the 2nd day. After seven days post-injury, a decrease in MPO content was observed in all groups, even in SLX and AS (p<0.05). On the 10th day, it showed the same profile as the 7th day. There was no significant difference between the groups in the studied times (p>0.05; Fig. 6).

Figure 6.

Quantification of the myeloperoxidase enzyme (MPO) of cutaneous abrasions treated topically with saline (S), antiseptic solution (AS), or latex serum (SLX) on the 2nd, 7th, and 10th days of follow-up. Values represent mean±standard error of mean (SEM). ANOVA statistical test, Bonferroni post-test. *Corresponds to a significant difference (p<0.05).

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